The present invention relates to compositions, methods and devices for the detection of infections resulting from xenogeneic transplants, particularly those caused by endogenous retroviruses. In particular, the present invention comprises compositions, devices and methods for the detection of porcine transplant materials, detection of which is necessary following xenotransplantation of porcine cellular products.
Currently, there are shortages of human organs, tissues and cells for transplantation into humans. Many patients awaiting a transplant die due to the lack of donor material. These shortages of human donor material suitable for allotransplantation, coupled with recent advances in transplantation immunology, have provided impetus for attempts to develop xenotransplantationxe2x80x94the therapeutic use of living animal tissues and organs in humans.
Although xenotransplants from animals such as pigs, baboons, and cows offer an unlimited source of organs and tissues, the therapeutic promise of xenotransplantation has not yet become widely accepted. The transplantation of simian organs and porcine cells and organs into humans has been reported, and progress has been encouraging enough to merit the beginning of limited clinical trials in the United States.
Pigs are among the primary animal species proposed as sources of xenografts. Xenotransplantation clinical trials involving porcine tissue being considered or underway include the perfusion through or implantation of whole liver preparations as a treatment for hepatic failure, the implantation of fetal neuronal tissue as a therapy for Parkinson""s disease, and the infusion or implantation of pancreatic islet cells as a treatment for diabetes mellitus. However, after xenotransplantation, detection and determination of persistence of the graft often involves expensive tests, such as CT scans, or invasive procedures such as a biopsy of the graft. When the graft is a diffuse transplantation of cells, sometimes none of these techniques are effective. What is needed are simple, non-invasive techniques for monitoring the presence and condition of the xenotransplantation materials.
In addition, concerns have been raised that the implantation of porcine tissue and/or cells into immune compromised humans may facilitate the transmission of new infectious agents to humans. The Public Health Service has therefore stressed the importance of proceeding with xenotransplantation clinical trials only after appropriate monitoring tests are available. What is especially needed are diagnostic tools that can determine the difference between transplanted xenograft materials and infectious agents that may originate from and may be present in the same xenotransplanted materials. Surveillance programs for new xenograft recipients need to be developed, and persons exposed to xenografts can be tested for evidence of graft persistence and possible xenogeneic infection.
Porcine tissues and cells are known to be infected with endogenous retroviruses. The genomes of all domesticated swine species tested thus far contain multiple integrated copies of an endogenous C-type retrovirus termed porcine endogenous retrovirus (PERV). The risks of transmission of known infectious agents may be reduced, or eliminated by procuring source animals from specific pathogen-free colonies. Therefore, one method for stopping the transmission of porcine endogenous viruses from xenotransplantation would be to harvest the transplantation materials from virus-free animals. However, this pre-transplant screening method cannot eliminate the porcine endogenous retrovirus (PERV), because the genome of these viruses is carried in the germ line of every pig. Pig PERV particles of type C morphology are released spontaneously by cell lines originating from a variety of pig tissues including kidneys, lymph nodes, testes and fallopian tubes. All known PERVs originate from healthy porcine tissues except for two known types, PERV-Shimozuma-1 and 38A-1 which are derived from porcine lymphomas.
The knowledge that PERV originating from both porcine cell lines and primary porcine lymphocytes can infect human cells in vitro has heightened safety concerns related to pig xenografts. Transmission of xenogeneic retroviral infections to xenograft recipients is of particular concern because retroviruses are known to result in life long persistent infections. Risks for xenogeneic infections may be significantly increased by the immunosuppressive therapies required to maintain graft function in human xenotransplant recipients. Currently, there are no rapid, inexpensive, and relatively noninvasive tests that can determine the presence of a xenotransplant and that may also determine that the patient is also free of a potential virus transmitted by the transplant. The current absence of the ability to detect the presence of PERV, which hampers the determination of whether PERV will infect humans exposed to porcine xenografts, and whether PERV will be transmitted secondarily among their contacts, has raised questions on the safety of pig-to-human transplantation, and threatens to delay progress in this therapeutic technology.
Accordingly, there is a need for rapid, sensitive, and specific compositions, methods and devices for detecting the presence and persistence of a xenotransplant in a xenotransplantation recipient and the status of xenograft survival. Further, there is a need to be able to determine the presence of any viruses or other infectious agents that might be present in the xenotransplant material, or transplant donor or the xenotransplant recipient. Additionally, there is a need to be able to determine graft status using noninvasive techniques, such as using body fluids from the transplant recipient. Such compositions, methods and devices would be particularly important for providing diagnostic and physiologic information for patients receiving porcine xenotransplants.
The present invention provides compositions, methods and devices for determining and monitoring xenograft integrity. The compositions, methods and devices are useful for determining or monitoring graft survival and rejection in recipients of xenografts. In addition the compositions, methods and devices are useful for differentiating between the presence of xenotransplant donor cells and the presence of xenogeneic endogenous retrovirus infections in a xenograft recipient. In particular, the compositions, methods and devices are useful for the differentiation between porcine cell microchimerism and true xenogeneic infection. Furthermore, the compositions, methods and devices are highly sensitive and can detect very low copy numbers of porcine or endogenous porcine viral sequences in the presence of 1 xcexcg of human DNA. The compositions, methods and devices are useful in defining the risks associated with the use of xenotransplantation, and in particular, porcine xenotransplantation. A particular advantage of the present invention is that body fluids of the transplant recipient can be used with the compositions, methods and devices of the present invention in determining the presence and persistence of the xenotransplant.
The compositions described herein comprise nucleic acid probes and primers useful for the amplification and detection of donor cells, such as probes for porcine mitochondrial DNA (mtDNA) and RNA, and for the presence of endogenous retroviruses. The methods described herein utilize the probes and primers with known amplification and detection techniques. The devices described herein employ the compositions and various components of the methods to facilitate the detection of the transplant and any endogenous virus. For example, the devices can be used to detect porcine cells, particularly porcine mtDNA and RNA, and PERV sequences.
Accordingly, it is an object of the present invention to provide methods and compositions for the rapid, sensitive, and specific detection of xenogeneic nucleic acids in body fluids and tissues of xenograft recipients.
Another object of the present invention is to provide compositions, methods and devices for monitoring xenograft survival and rejection.
Yet another object of the present invention is to provide compositions, methods and devices for differentiating between the presence of animal donor cells and infections such as those with endogenous retroviruses.
Another object of the present invention is to provide compositions, methods and devices that employ biological fluids such as serum or plasma for use in biological assays.
Still another object of the present invention is to provide compositions, methods and devices for screening for infectious agents inherently present in pig xenografts, by detecting pig cell-specific markers in biological products such as pig factor VIII, porcine insulin and porcine heparin.
It is another object of the present invention to provide compositions, methods and devices for detecting the presence of endogenous retroviruses in transplant material, including tissues, organs and cells.
Still another object of the present invention is to provide compositions, methods and devices for determining the zoonotic potential and tissue tropism of endogenous retroviruses.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.